Prevalence and diversity of Wolbachia bacteria infecting insect pests of stored products

Wolbachia are common bacterial symbionts of insects. Because infections of these bacteria can alter the reproduction and biology of the host, there is interest in the potential use of Wolbachia to control populations of pest species. To advance this research, we screened 38 species (88 populations from 16 countries) of stored-product insect pests for Wolbachia infections. Infections were detected in nine species of Coleoptera (Anobiidae – Lasioderma serricorne (Fabricius), Stegobium paniceum (Linnaeus); Curculionidae – Sitophilus oryzae (Motschulsky), Sitophilus zeamais Motschulsky; Dermestidae – Attagenus unicolor (Brahm), Dermestes lardarius Linnaeus; Silvanidae – Oryzaephilus mercator (Fauvel), Oryzaephilus surinamensis (Linnaeus); Tenebrionidae – Tribolium confusum Jaquelin Du Val), one species of Hymenoptera (Trichogrammatidae – Trichogramma deion Pinto & Oatman), and one species of Lepidoptera (Pyralidae – Ephestia kuehniella Zeller). Phylogenetic analyses based on wsp gene sequences identified all Wolbachia isolates as members of Supergroups A or B. Additional sequences for the genes gatB, coxA, hcpA, fbpA and ftsZ were obtained for use in multilocus sequence typing (MLST) to identify the sequence types to which the isolates belonged. Our results identify five new Wolbachia-insect associations and support previous findings that infections of Wolbachia in stored-product insect pests are members of Supergroups A or B.     

Isolation and characterization of Listeria monocytogenes in Chinese food obtained from the central area of China

The objective of this study was to investigate the prevalence and characteristics of Listeria monocytogenes isolated from Chinese food, including frozen dumplings, flavored raw meat, roasted meat, braised meat, and a cold vegetable dish with sauce. A total of 900 food samples were collected from supermarkets, open-air markets, and delicatessens in three large cities in the central area of China to examine the presence of L. monocytogenes; 21 (2.3%) of the samples were positive for this pathogen. Among the different samples, braised meat showed the highest L. monocytogenes detection rate (4.4%). Samples obtained from delicatessens showed a much higher L. monocytogenes contamination rate (8.3%) than those from open-air markets (6.7%) or supermarkets (0%). Multilocus sequence typing (MLST) analysis indicated that the 21 bacterial isolates belonged to 12 ST subgroups. ST5 was the largest and contained 7 isolates (33.3%); it was followed by ST474, ST121 and ST9 (each containing 2 isolates [10.5%]). Antibiotic susceptibility analysis showed that the 21 L. monocytogenes isolates were thoroughly resistant to cefoxitin but highly susceptible to doxycycline and ciprofloxacin. The presence of 10 virulence genes was evaluated by PCR, which showed that inlA, inlC, inlJ, prfA, hlyA, and plcB were present in all isolates and that inlB, actA, plcA and iap were present in 71.4–90.5% of the isolates. This study provides a useful reference for risk assessment and control of L. monocytogenes contamination in Chinese food and for the treatment of clinical listeriosis.     

Phenotypic Characterization and Genotypic Subtyping of Salmonella enterica Serovars Enteritidis and Gallinarum Isolated from Human and Poultry-Related Samples

This study aimed to assess the serotypes, antibiotic susceptibility, and molecular subtyping of Salmonella enterica isolated from food products and human patients with gastroenteritis. A total of 59 isolates were investigated, and the results revealed a predominance of S. Enteritidis with 57.63% (34/59) S. Gallinarum held second with 15.62% (5/32) of total food borne. While, isolates from humans showed 18.51% (5/27) of S. Typhimurium. High level of resistance to nalidixic acid was noted among food strains and 35.29% of human isolates were found to be multidrug resistant. Eight representative isolates were subtyping using three molecular approaches, ribotyping, Multilocus sequence typing (MLST) and Multiple-locus variable number tandem repeat analysis (MLVA). MLST showed three sequence types corresponding to two clonal complexes, (ST-78, CC-4) for S. Gallinarum, (ST-11, CC-4) for S. Enteritidis and (S-367, CC-37) for S. Cerro. While, MLVA generated six different profiles targeting nine loci for S. Enteritidis and S. Gallinarum.     

华南地区食品中非O157致泻大肠杆菌分布特点及分型研究

为深入了解华南地区食品中非O157致泻大肠杆菌(DEC)的污染分布情况和特点,本研究随机采集了该地区12个城市的食品样品,参考GB/T 4789.36-2003方法进行检测,并利用多重PCR对DEC进行了分子鉴定。另外,分别采用MLST分型方法和药敏纸片法对DEC菌株的遗传特性和耐药性进行了分析。结果表明,1000份样品中有164份检出DEC,总污染率高达16.4%。肉类和水产品污染较严重。在五种DEC中,EPEC检出率最高(8.0%),其次为ETEC(6.2%),EIEC(3.4%)和STEC(0.4%)。样品中共分离到207株DEC。MLST分型产生了58种ST型,其中44个为已报道的ST型,14个为数据库中新的ST型。聚类分析表明这些菌株共有8个克隆复合物(CC),CC10为其中最大的一个。DEC菌株对四环素、复方新诺明、氨苄西林、头孢噻吩和氯霉素等具有高抗性。有关部门应加强非O157致泻大肠杆菌的监控,减少食源性疾病的发生。     

Short communication: Occurrence of methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci in dairy goat herds in Ohio,United States

In light of the scarcity of information about the occurrence and epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MRCNS) in small ruminants in general, and particularly dairy goats, we launched this limited-scope study. The findings reported here show the detection of MRSA and MRCNS in goat milk and teat skin samples from dairy goat herds in the state of Ohio. A total of 120 milk samples and 120 teat-swab samples were collected from 5 farms. After conventional isolation and phenotypic characterization of the staphylococci colonies, bacterial isolates were tested by PCR assay targeting the genes nuc to identify Staphylococcus aureus and mecA to detect MRSA and MRCNS. The clonal complexes of MRSA isolates was also determined by multiloccus sequence typing. Fifteen (6.2%) positive S. aureus samples were found in this study: 9 from milk and 6 from teat skin samples. Four (2%) MRSA isolates were detected and, using multiloccus sequence typing genotyping, these were designated to clonal complexes CC133 (n = 2; milk samples) and CC5 (n = 2; teat skin). Three (1.25%) coagulase-negative staphylococci isolates from the teat skin also harbored the mecA gene. Although, the MRSA isolated from milk samples is not a typical human-associated lineage, the CC5 clone isolated from teat skin is a common and widespread clonal complex associated with humans, suggesting that this extramammary niche could be a relevant reservoir of methicillin-resistant staphylococci. Furthermore, the fact that 75% of MRSA were recovered from 1 farm showing poor hygiene practices strengthens the hypothesis that good hygiene practices could be useful to prevent persistence and spread of MRSA at a farm level.     

Specific populations of the yeast Geotrichum candidum revealed by molecular typing

Geotrichum candidum is a ubiquitous yeast and an essential component in the production of many soft cheeses. We developed a multilocus sequence typing (MLST) scheme with five retained loci (NUP116, URA1, URA3, SAPT4 and PLB3) which were sufficiently divergent to distinguish 40 sequence types (STs) among the 67 G. candidum strains tested. Phylogenetic analyses defined five main clades; one clade was restricted to environmental isolates, three other clades included distinct environmental isolates and dairy strains, while the fifth clade comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or the dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group of specialized European G. candidum strains. In addition, we developed a polymerase chain reaction inter‐long terminal repeat scheme, a fast and reproducible random amplification of polymorphic DNA‐like method for G. candidum, to type the closely related dairy strains, which could not be distinguished by MLST. Overall, our findings distinguished two types of dairy strains, one forming a homogeneous group with little genetic diversity, and the other more closely related to environmental isolates. Neither regional nor cheese specificity was observed in the dairy G. candidum strains analysed. This present study sheds light on the genetic diversity of both dairy and environmental strains of G. candidum and thus extends previous characterizations that have focused on the cheese isolates of this species. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of Automated Ribotyping,spa Typing,and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer^® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods.     

An Improved MLVF Method and Its Comparison with Traditional MLVF,spa Typing,MLST/SCCmec and PFGE for the Typing of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen, causing considerable morbidity and mortality. During the last 20 years, a variety of genotyping methods have been introduced for screening the prevalence of MRSA. In this study, we developed and evaluated an improved approach capillary gel electrophoresis based multilocus variable-number tandem-repeat fingerprinting (CGE/MLVF) for rapid MRSA typing. A total of 42 well-characterized strains and 116 non-repetitive clinical MRSA isolates collected from six hospitals in northeast China between 2009 and 2010 were tested. The results obtained by CGE/MLVF against clinical isolates were compared with traditional MLVF, spa typing, Multilocus sequence typing/staphylococcal cassette chromosome mec (MLST/SCCmec) and pulse field gel electrophoresis (PFGE). The discriminatory power estimated by Simpson’s index of diversity was 0.855 (28 types), 0.855 (28 patterns), 0.623 (11 types), 0.517 (8 types) and 0.854 (28 patterns) for CGE/MLVF, traditional MLVF, spa typing, MLST/SCCmec and PFGE, respectively. All methods tested showed a satisfied concordance in clonal complex level calculated by adjusted Rand’s coefficient. CGE/MLVF showed better reproducibility and accuracy than traditional MLVF and PFGE methods. In addition, the CGE/MLVF has potential to produce portable results. In conclusion, CGE/MLVF is a rapid and easy to use MRSA typing method with lower cost, good reproducibility and high discriminatory power for monitoring the outbreak and clonal spread of MRSA isolates.     

Survival of poultry-derived Campylobacter jejuni of multilocus sequence type clonal complexes 21 and 45 under freeze,chill, oxidative,acid and heat stresses

The application of multilocus sequence typing (MLST) for studying Campylobacter jejuni diversity reveals that MLST clonal complex (CC) 21 and CC-45 occupies significant proportion in the diverse population of C. jejuni. These two complexes are ecologically abundant and represent an interesting subpopulation for studying C. jejuni survival under different stress conditions. In the present study we characterize and compare 19 C. jejuni strains assigned to CC-21 and CC-45, isolated from chicken meat, based on laboratory stress models maintained in Muller-Hinton broth. Model conditions were mimicking freeze, chill, oxidative, acid and heat stresses. Results show that survival patterns varied between the strains. C. jejuni strains of CC-21 survived significantly better than C. jejuni strains of CC-45 under heat (P value = 0.022) and chill (P value = 0.001) stress models. On the other hand, C. jejuni strains of CC-45 showed significantly better survival compared to C. jejuni strains of CC-21 in response to oxidative (P value = 0.003) and freeze (P value = 0.021) stress models. C. jejuni strains assigned to the founder ST-45 showed significantly better survival (P value = 0.017) under heat stress model compared to their ancestral sequence types. However, an association between survival fitness and the diversification of a clonal group cannot be demonstrated directly from the obtained results. In conclusion, findings of the present study show that genotypic variations of C. jejuni might play a role in enabling certain lineages to be selected when encountering adverse and stressful environments. In future stress response studies, it is recommended to consider the effect of genotypic diversity among C. jejuni strains as that might bias the experimental findings.     

A 12-month longitudinal study of Listeria monocytogenes contamination and persistence in pork retail markets in China

Listeria monocytogenes is a foodborne pathogen frequently isolated from raw pork meat. This study was designed to investigate the prevalence and molecular characteristics of L. monocytogenes in raw pork from open markets in China. The survey was conducted monthly over a 12-month period in Zigong, China. L. monocytogenes was isolated from 262 of 1641 samples collected (16.0%) including minced meat samples (131/608, 21.5%), pork pieces samples (111/857, 13.0%) and environmental swabs (20/176, 11.4%). The isolation rates in spring and winter were significantly higher than those in summer and autumn (X² = 68.85, P < 0.05). All isolates were subjected to serotyping, multi-locus sequence typing (MLST) and AscI pulsed-field gel electrophoresis (PFGE). The 262 isolates were subtyped into five serotypes: 1/2b (43.1%), 1/2c (35.5%), 1/2a (19.1%), 4b (1.1%), 3a (1.1%); 20 sequence types (STs) with four most frequent STs, being ST9 (35.9%), ST87 (19.8%), ST3 (16.0%) and ST8 (14.1%); and 39 pulsotypes (PTs) with PT4 (26.3%), PT30 (14.5%) and PT11 (12.6%) being most frequent. Two primary pulsotypes from pork pieces were previously isolated from clinical listeriosis cases in the local hospitals. The six markets from different districts differed in the level of contamination and strain types. Persistent contamination of L. monocytogenes was found in the markets especially in meat mincers, which were found to be one likely source of continuous cross contamination. These findings will help develop strategies to reduce L. monocytogenes contamination in open markets for better public health control and prevention of foodborne L. monocytogenes infections.